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Cystinosis research foundation, 6 months progress report UNRAVELLING THE MECHANISMS OF CYSTEAMINE TOXICITY IN PATIENTS WITH CYSTINOSIS Martine Besouw, MD, PhD student Elena Levtchenko, MD, PhD, principal investigator Department of Pediatric Nephrology, University Hospital Gasthuisberg Herestraat 49, 3000 Leuven, Belgium 1 Background Nephropathic cystinosis is an autosomal recessive metabolic disorder caused by cystine accumulation within lysosomes. An amino thiol cysteamine is currently the only available treatment, depleting lysosomal cystine and postponing renal and extra-renal organ damage in cystinosis. During recent years the strategy to administer the highest tolerable doses of cysteamine was challenged by the development of serious adverse events consisted of skin lesions (striae, vascular tumors on the elbows), bone pain, muscular weakness and vascular complications in a few patients, treated with high cysteamine doses. Histological analysis of the skin in one patient demonstrated irregularities of the elastin network and collagen fiber caliber, resembling lesions described in Ehlers-Danlos syndrome (EDS) and endothelial proliferation disrupting underlying extracellular matrix. Specific aims Because only a small proportion of cystinosis patients, treated with high cysteamine doses, developed the above-mentioned symptoms, we hypothesized that these patients might have a genetic susceptibility to cysteamine. In this project we aim to compile a risk profile, using the combination of genetic make-up and molecular expression patterns of relevant markers using genomic DNA and cultured skin fibroblasts of cystinosis patients suffering from cysteamine adverse events, compared to the patients treated with high cysteamine doses without the above-mentioned symptoms and those of healthy subjects. The availability of biomarkers for cysteamine endurance would allow: a) to identify patients at risk, requiring extra medical attention and eventually cysteamine dose reduction and b) to monitor early phases of cysteamine hyperresponsiveness. Key-objectives/work plan A. To collect full information on patients reported with the above-mentioned symptoms. B. To investigate whether or not there are differences in cysteamine plasma levels between patients with and patients without severe adverse events. C. To analyze genetic variations in genes involved in classical EDS. D. To investigate the effects of a range of cysteamine concentrations on different human cell lines. Results A. Clinical information was obtained from the patients’ physicians and is summarized in table I. A manuscript is currently being drafted. 2 Patient Sex GFR (mL/min/ 1.73 m2) CTNS mutation Age at onset cystinosis treatment Concomitant medications 1 Male 87 Unknown 2 years, 1 month L-thyroxine L-carnitine Potassium citrate 2 Male 161 Unknown 1 year, 1 month Unknown 3 Female 25 Unknown 9 months Potassium Somatotropin Folic acid Riboflavin (Vit B2) Iron Pyridoxine (Vit B6) Indomethacin Somatotropin Epoietin beta 4 5 Male Male 62 30 c.18-21 del GACT 11 months c.18-21 del GACT Unknown 1 year, 1 month 6 Male 101 57kb del 57kb del 7 years, 8 months 7 Male 67 Unknown 2 years, 2 months L-thyroxine Somatotropin L-carnitine Calcitriol Sodium chloride Epoietin beta Phosphate Folic acid Sodium bicarbonate citrate Phosphate Hydrochlorothiazide Potassium chloride Alphacalcidiol Esomeprazol Alphacalcidol Potassium bicarbonate Sodium potassium phosphate L-carnitine Somatotropin Iron Potassium Phosphoric acid Alphacalcidiol Nicardipine Citrate Indomethacin Sodium bicarbonate 8 Male 73 Unknown 9 months Potassium chloride bicarbonate Sodium bicarbonate Alphacalcidiol Table I. Clinical information of all reported patients with Ehlers-Danlos like syndrome B. Cysteamine plasma curves were made in 2 of the above-mentioned patients (patient 3 and 6) and 5 patients without similar events after the intake of 15 mg/kg of cysteamine base (figure 1). So far no difference was found between these 2 types of patients. 3 Figure 1. Cysteamine plasma curves of 5 patients without events (control patients) and 2 patients with Ehlers-Danlos like syndrome (patient 3 and 6). C. Blood samples were obtained from 3 patients with and 5 patients without Ehlers-Danlos like syndrome . They will be screened for aberrations in the genes involved in classical EDS (COL5A1, COL5A2, COL1A1, COL1A2, TNXB). The DNA and fibroblasts samples of the other patients are required. D. We investigated the response of human endothelial cells (HUVEC, human umbilical vein endothelial cell line, commercially available at Lonza Group Ltd, Basel, Switzerland) to different concentrations of cysteamine. Cysteamine was added to the medium in concentrations of 0.03 mM, 0.1 mM, 0.3 mM, 1.0 mM, 3.0 mM and 10 mM. One group of control cells received no cysteamine. All these seven concentrations were administered during 1, 6 and 24 hours. After that time, the viability and proliferation of the cells was investigated in order to estimate the effects of cysteamine on normal human endothelial cells using the commercial available WST1 Rapid Cell Proliferation Kit (Clontech). The results are pending. 4